GWAS of Thermus thermophilus
Overview Thermus thermophilus is an aerobic oblique heterotroph, which is consistently found in environments with temperatures as high as 85°C. This specific bacterium shows high transformation competence, and is therefore amenable to genetic manipulation. T. thermophilus has become a model organism is structural biology. Many of its enzymes have been crystallized, and their structures analyzed using x-ray crystallography. Subjects for research have included the structural basis for protein thermostability, and the adaption strategies for survival at high temperatures. The stability of thermostable enzymes and their resistance to denaturation by physical or chemical factors are considerable advantages in the industrial process. Various enzymes of the thermus species have been used in biotechnological applications such as DNA polymerase in PCR reactions. Other applicable uses for thermostable enzymes include starch processing, organic synthesis, waste treatment, pulp and paper manufacturing, and amino acid and vitamin synthesis. The genome of T. thermophilus HB27 was sequenced using the random shotgun method. After analyzing the genome, the focus was directed to identifying genes of potential biotechnological value. Sequencing Methods To sequence the genome of T. thermophilus HB27, the bacterium was grown in culture from an isolated single cell. Once a sufficient number of cells was obtained, the total genomic DNA was extracted and sheared. Several shotgun libraries were created using size fractions ranging for 1-3kbp. A cosmid library was then constructed from sau3AI partially digested genomic DNA cloned in the cosmid vector SuperCos1. Insert ends of the recombinant plasmids were then sequences using ABI Prism 377 DNA sequencers. A coverage of 9.1 fold was determined after the assembly of about 28,000 sequences. The remaining overlapping DNA sequences were ordered according to a previously determined physical map containing the locations of 61 genes from T. thermophilus and other Thermus strains. Problems associated with misassembled regions and repetitive sequences was solved through application of PCR based techniques and primer walking on recombinant plasmids. A mean error rate of <1 per 10,000 bases was achieved using this process. Metabolic characteristics with biotechnological applications Through genomic sequencing, it was found that T.thermophilus contains all of the genes necessary for vitamin B12 synthesis. Vitamin B12 is essential in the maintenance and regulation of the brain and nervous system, as well as formation of red blood cells. Therefore, this organism could potentially be exploited as a producer of vitamin B12 in thermophilic conditions. The chromosomal encoded genes are disperse, and encode proteins involved in the pathway that convert aminolevulinate into uroporphyrinogen III, which are precursors of all tetrapyrrols. Tetrapyrrols are a class of molecules that comprise heme, the core molecule in hemoglobin. It is believed that the capability of T. thermophilus to synthesize tetrapyrrols has evolved independently from the specialization vitamin B12 formation. Combining these two functional genes however could prove to be extremely beneficial in a pharmaceutical sense. It was also found that T. thermophilus has the capability to undergo carotenoid biosynthesis. Carotenoids are a class of natural pigments that are of interest as food colorants and nutrient supplements. There is currently an ongoing debate about their potential cancer preventative properties. T. thermophilus produces a yellow carotenoid pigment, referred to chemically as thermozeaxanthins. The terminal steps of carotenoid synthesis is encoded on the plasmid, where as precursors of synthesis are accompanied by enzymes that are encoded on the chromosome. Although plasmid-encoded carotenoid biosynthesis genes seem to not be organized in a single gene cluster, their proximity is apparent and therefore still has the potential to be exploited industrially. References [[wikipedia:Carotenoid|1. "Carotenoid." Wikipedia. Wikimedia Foundation, 27 Oct. 2014. Web. 02 Nov. 2014.]] [http://www.nature.com/nbt/journal/v22/n5/pdf/nbt956.pdf 2. Thermophile, The Genome Sequence of the Extreme Thermophile, Thermus Thermophilus Anke Henne, Holger Brüggemann, Carsten Raasch, Arnim Wiezer, Thomas Hartsch, Heiko Liesegang, (n.d.): n. pag. Nature.com. Web. 2 Nov. 2014.]